Expression Of SNAP-25 Research Articles

Accelerated ovarian failure results in brain alterations related to Alzheimer's disease that are not recovered by high-intensity interval training in mice.

The menopausal decline in ovarian estrogen production is thought to increase the risk of Alzheimer's disease; however, this link requires further investigation. The chronological development of this connection is not well defined because of the lack of animal models that recapitulate the time course of menopause. This study characterized the cognitive and neuronal effects of the 4-vinylcyclohexene diepoxide (VCD) model of ovarian failure in female mice and assessed whether high-intensity interval training (HIIT) would attenuate impairments. Female mice were injected with VCD for 15 days. Novel object recognition tests (NORT) were conducted during (perimenopause) and after (menopause) ovarian failure (n=7). HIIT was initiated in menopause and mice underwent NORT testing after 2 and 8weeks of HIIT (n=5). VCD mice had a lower discrimination index, and lower SNAP25 and NeuN expression in perimenopause. HIIT did not recover memory in VCD mice. Neuronal changes arise early in the perimenopausal transition and HIIT did not improve recognition memory when initiated in menopause. The menopausal decline in ovarian estrogen production increases the risk of Alzheimer's disease (AD). The chronological development of this connection is not well defined because of the lack of animal models that recapitulate the time course of menopause. 4-vinylcyclohexene diepoxide (VCD)-induced ovarian failure provides a model that simulates the average human experience in the transition from perimenopause to menopause. We demonstrate that cognitive and biochemical effects related to AD pathology are present from the earliest available timepoint in perimenopause in VCD mice. This work highlights the importance of examining the time course in the progression to menopause and the use of VCD as a model to investigate changes in the brain.

AnMei decoction ameliorates cognitive impairment in rats with chronic sleep deprivation by mitigating hippocampal neuroinflammation and restoring synaptic architecture

Significance of ethnopharmacologyAnMei Decoction (AMD) is a renowned herbal prescription that has been widely demonstrated to have positive therapeutic effects on sleep disorders, depression, and cognitive impairments. However, the molecular mechanisms underlying AMD's resistance to sleep deprivation-induced cognitive impairment remain to be further investigated. Research objectiveTo clarify whether AMD may alleviate neuroinflammation by inhibiting NLRP3/Caspase1 signaling pathway and repair neuronal damage by regulating BDNF/TrkB pathway, thereby improving cognitive dysfunction in rats with chronic sleep deprivation. Materials and methodsLC-MS/MS was used to detect the active components in AMD. After behavioral tests, HE staining, Nissl staining, immunofluorescence, immunohistochemistry, transmission electron microscopy, and Golgi staining were performed to assess the effects of AMD on chronic sleep deprivation. Western blot was used to detect the expression of hippocampal proteins NLRP3, Caspase-1, BDNF, p-TrkB, TrkB, Bax, Bcl-2, GAP43, PSD95, SNAP25, SYN, STX1A, and VAMP2. Hippocampal transcriptome sequencing was employed to observe differentially expressed genes after AMD intervention. ResultsA total of 15 active components were identified from the AMD extract. AMD effectively improved the exploration and learning and memory abilities of sleep-deprived rats. AMD reduced neuroinflammation by inhibiting the NLRP3/Caspase-1 pathway and repaired neuronal damage by regulating the BDNF/TrkB pathway. Simultaneously, AMD upregulated the expression of BDNF, p-TrkB, Bcl-2, GAP43, PSD95, SNAP25, SYN, STX1A, and VAMP2 proteins and inhibited the expression of NLRP3, Caspase-1, and Bax proteins. Analysis of GO and KEGG pathway enrichment for the differentially expressed inflammation-related pathways may be involved in the therapeutic mechanism of AMD on sleep deprivation. ConclusionAMD can effectively inhibit the NLRP3/Caspase1 signaling pathway to alleviate neuroinflammation, regulate the BDNF/TrkB pathway to maintain hippocampal neuronal viability, repair synaptic structural damage, and improve cognitive impairment in the sleep deprivation model.

Carbohydrate response element-binding protein (ChREBP) mediates decreased SNAP25 expression in islets from diabetic Goto-Kakizaki (GK) rats.

SNAP25 plays an essential role in the glucose-stimulated insulin secretion (GSIS) of pancreatic β-cells. Carbohydrate response element-binding protein (ChREBP) is an important transcription factor in β-cells and, in this study, we aimed to explore whether ChREBP regulates SNAP25 expression in β-cells. We show that diabetic Goto-Kakizaki (GK) rats exhibited impaired insulin secretion and hyperglycemia, along with decreased SNAP25 expression and ChREBP phosphorylation in islets. SNAP25 knockdown decreased GSIS in β-cells, while SNAP25 overexpression increased GSIS in β-cells. Activation or overexpression of ChREBP led to reduced SNAP25 expression and subsequent suppression of GSIS. Conversely, ChREBP knockdown mitigated the reduction in SNAP25 expression caused by high glucose. Mechanistically, the activation of ChREBP by high glucose increased its occupancy and decreased the level of H3K4me3 at the Snap25 promoter. Our findings reveal the novel regulatory mechanisms of SNAP25 expression in β-cells and suggest that SNAP25 may be involved in the regulation of β-cell secretory function controlled by ChREBP.

Boldine Promotes Recovery of Function After Motor-Incomplete Spinal Cord Injury in Mice

Membrane channels such as connexins (Cx) and pannexins (Panx) are permeable to calcium ions and other small molecules such as ATP and glutamate. Release of ATP and glutamate through these channels is a key mechanism driving tissue response to traumas such as spinal cord injury (SCI). Boldine, an alkaloid isolated from the Chilean boldo tree, blocks both Cx hemichannels (HC) and Panx. To test if boldine could improve function after SCI, boldine or vehicle was administered to treat mice with a moderate severity contusion-induced SCI. Boldine led to greater spared white matter and increased locomotor function as determined by the Basso Mouse Scale and horizontal ladder rung walk tests. Boldine treatment reduced immunostaining for markers of activated microglia (Iba1) and astrocytic (GFAP) markers while increasing that for axon growth and neuroplasticity (GAP-43). RT-qPCR studies showed that boldine treatment reduced expression of chemokine Ccl2, cytokine IL-6 and microglial gene CD68, while increasing expression of the neurotransmission genes Snap25 and Grin2b, and Gap-43. Bulk RNA sequencing revealed that boldine modulated a large number of genes involved in neurotransmission in spinal cord tissue just caudal from the lesion epicenter at 14 days after SCI. Numbers of genes regulated by boldine was much lower at 28 days after injury. Furthermore, physical activity through treadmill training together with boldine showed an additive effect in recovery of function after SCI. Overall, our results indicate that boldine treatment ameliorates injury and spares tissue to increase locomotor function. DOD SCIRP SC170315; VA RR&D Service Grant B-2020-C; NIH GM54508 and GM137056. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Glial-Neuronal Crosstalk in the Ageing Murine Nodose Ganglion

Background: Viscerosensory stimuli are transmitted from the peripheral nervous system to the central nervous system for processing, integration, and adequate motor response. Proper signal transmission is guarded by bidirectional interaction between neurons and glia and their dysfunction has been associated with sensory deficits in both aging and visceral disorders. However, compared to the central nervous system, the bidirectional interactions and pathways of communication between glia and neurons remain relatively unknown. The nodose ganglion (NG) houses the cell bodies of visceral sensory nerves, traveling from the periphery, via the vagus nerve, to the central nervous system. Autonomic remodeling in this ganglion is associated with many of the visceral diseases that increase in incidence with age. Given the increasing recognition of the importance of glia in neuronal function and neurotransmission, understanding their inter-cell dynamics are vital to appropriately interpret the neurophysiology of aging. Objectives: This study aimed to better understand subtypes of neurons and glia most involved in neuro-glial communication, the underlying genetic pathways involved in this crosstalk, and how these pathways change with age. Methods: Single-cell RNA sequencing (scRNAseq) was performed on NG of young (12 weeks) and old (16 months) mice (C57BL/6; N = 6 for both groups). Distinct satellite glial cell (SGC) and neuronal populations were clustered based on transcriptomic similarities using dimensionality reduction. Marker gene analyses were used for cell-type identification. Specific pathways responsible for glial-glial, neuron-neuron, and glial-neuronal cross talk, including secreted signaling, extracellular-matrix receptor interactions, and cell-cell contact, were assessed between the different glial and neuronal sub-clusters in young and aged NG. The R-packages Seurat and CellChat were used for cell clustering and cell-cell communication signaling pathway identification, respectively. Results: SGC ( N = 6835 cells total) were identified by high expression of glial-specific transcripts, S100b and Fabp7. Neurons ( N = 2027 neurons total) were identified by expression of Tubb3, Snap25, and Uchl1. Five distinct glial clusters and six distinct neuronal clusters were identified by expression of known functional marker genes. While neuron-to-neuron communication was limited, significant communication within glial subtypes and between glia and sensory neurons was noted, with residential SGCs being most central to this network. Residential SGC interactions were noted most significantly for excitatory, multimodal neuronal subtypes ( Vglut2+, TRPV1/2+, Piezo1/2+). The primary pathways driving this communication were trophic pathways (e.g., vascular endothelial growth factor , Tenascin C, and Thrombospondin-1). Surprisingly, while glia-glia signaling did not change with aging, glial-neuronal communication seemed to increase. Conclusion: These data suggest that, while there is limited communication between neurons in the nodose ganglia, there is extensive glial-glial and glial-neuronal communication. Resident SGCs and excitatory multimodal neurons, which are known to be critical in sensory neurotransmission, are central to this crosstalk. Aging was associated with increased glial-neuronal crosstalk, though the cause and effect of this increased glial-neuronal communication with age requires further investigation. Dr. Vaseghi is supported by NIH R01HL148190 and AHA 970217. VvW is supported by the NWO Rubicon grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Endoplasmic reticulum stress impedes regulated secretion by governing key exocytotic and granulogenic molecular switches.

Dense core vesicles (DCVs) and synaptic vesicles are specialised secretory vesicles in neurons and neuroendocrine cells, and abnormal release of their cargo is associated with various pathophysiologies. Endoplasmic reticulum (ER) stress and inter-organellar communication are also associated with disease biology. To investigate the functional status of regulated exocytosis arising from the crosstalk of a stressed ER and DCVs, ER stress was modelled in PC12 neuroendocrine cells using thapsigargin. DCV exocytosis was severely compromised in ER-stressed PC12 cells and was reversed to varying magnitudes by ER stress attenuators. Experiments with tunicamycin, an independent ER stressor, yielded similar results. Concurrently, ER stress also caused impaired DCV exocytosis in insulin-secreting INS-1 cells. Molecular analysis revealed blunted SNAP25 expression, potentially attributed to augmented levels of ATF4, an inhibitor of CREB that binds to the CREB-binding site. The effects of loss of function of ATF4 in ER-stressed cells substantiated this attribution. Our studies revealed severe defects in DCV exocytosis in ER-stressed cells for the first time, mediated by reduced levels of key exocytotic and granulogenic switches regulated via the eIF2α (EIF2A)-ATF4 axis.

GLP-1 signaling-regulated SNAP25 is involved in improved insulin secretion in diabetic GK rats after Roux-en-Y gastric bypass surgery.

Roux-en-Y gastric bypass surgery (RYGB) improves glucose-stimulated insulin secretion (GSIS) in type 2 diabetes (T2D) patients. SNAP25 plays an essential role in GSIS. Clinical studies indicate that enhanced GLP-1 signaling is an important contributor to the improved β-cell function in T2D. We aimed to explore whether GLP-1-regulated SNAP25 is involved in the enhanced secretory function of β-cells in diabetic Goto-Kakizaki (GK) rats after RYGB. RYGB or sham surgery was conducted in GK rats. mRNA and protein expression of SNAP25 was assessed by qPCR and Western blot, respectively. Occupancy of CREB and acetyltransferase CBP and acetylation of histone H3 (ACH3) at the Snap25 promoter were determined using ChIP assay. RYGB led to increased SNAP25 expression and CREB phosphorylation in islets from GK rats. Increased SNAP25 improved GSIS in β-cells cultured in high glucose conditions. Consistent with increased plasma GLP-1 after RYGB, GLP-1R agonist exendin4 increased SNAP25 expression and CREB phosphorylation in β-cells. Mechanistically, exendin4 promoted the recruitment of CREB and CBP, thereby increasing ACH3 at the Snap25 promoter. Consistently, inhibition of CBP attenuated the effect of exendin4 on SNAP25 expression. Furthermore, the knockdown of SNAP25 diminished the increase of GSIS potentiated by chronic GLP-1 culture in INS-1 832/13 cells. Our findings unravel the novel mechanisms of RYGB-enhanced SNAP25 expression in β-cells, and SNAP25 may contribute to the improved β-cell secretory function induced by RYGB.

Fat mass and obesity-associated (FTO) gene is essential for insulin secretion and β-cell function: In vitro studies using INS-1 cells and human pancreatic islets

AimsIn this study, we investigated the role of the FTO gene in pancreatic β-cell biology and its association with type 2 diabetes (T2D). To address this issue, human pancreatic islets and rat INS-1 (832/13) cells were used to perform gene silencing, overexpression, and functional analysis of FTO expression; levels of FTO were also measured in serum samples obtained from diabetic and obese individuals. ResultsThe findings revealed that FTO expression was reduced in islets from hyperglycemic/diabetic donors compared to normal donors. This reduction correlated with decreased INS and GLUT1 expression and increased PDX1, GCK, and SNAP25 expression. Silencing of Fto in INS-1 cells impaired insulin release and mitochondrial ATP production and increased apoptosis in pro-apoptotic cytokine-treated cells. However, glucose uptake and reactive oxygen species production rates remained unaffected. Downregulation of key β-cell genes was observed following Fto-silencing, while Glut2 and Gck were unaffected. RNA-seq analysis identified several dysregulated genes involved in metal ion binding, calcium ion binding, and protein serine/threonine kinase activity. Furthermore, our findings showed that Pdx1 or Mafa-silencing did not influence FTO protein expression. Overexpression of FTO in human islets promoted insulin secretion and upregulated INS, PDX1, MAFA, and GLUT1 expression. Serum FTO levels did not significantly differ between individuals with diabetes or obesity and their healthy counterparts. ConclusionThese findings suggest that FTO plays a crucial role in β-cell survival, metabolism, and function and point to a potential therapeutic utility of FTO in T2D patients.

SNAP-25, but not SNAP-23, is essential for photoreceptor development, survival, and function in mice

SNARE-mediated vesicular transport is thought to play roles in photoreceptor glutamate exocytosis and photopigment delivery. However, the functions of Synaptosomal-associated protein (SNAP) isoforms in photoreceptors are unknown. Here, we revisit the expression of SNAP-23 and SNAP-25 and generate photoreceptor-specific knockout mice to investigate their roles. Although we find that SNAP-23 shows weak mRNA expression in photoreceptors, SNAP-23 removal does not affect retinal morphology or vision. SNAP-25 mRNA is developmentally regulated and undergoes mRNA trafficking to photoreceptor inner segments at postnatal day 9 (P9). SNAP-25 knockout photoreceptors develop normally until P9 but degenerate by P14 resulting in severe retinal thinning. Photoreceptor loss in SNAP-25 knockout mice is associated with abolished electroretinograms and vision loss. We find mistrafficked photopigments, enlarged synaptic vesicles, and abnormal synaptic ribbons which potentially underlie photoreceptor degeneration. Our results conclude that SNAP-25, but not SNAP-23, mediates photopigment delivery and synaptic functioning required for photoreceptor development, survival, and function.

Identification of Skp1 as a target of mercury sulfide for neuroprotection.

Mercury sulfide (HgS) exerts extensive biological effects on neuronal function. To investigate the direct target of HgS in neuronal cells, we developed a biotin-tagged HgS probe (bio-HgS) and employed an affinity purification technique to capture its target proteins. Then, we identified S-phase kinase-associated protein 1 (Skp1) as a potential target of HgS. Unexpectedly, we discovered that HgS covalently binds to Skp1 through a "Cys62-HgS-Cys120" mode. Moreover, our findings revealed that HgS inhibits the ubiquitin-protease system through Skp1 to up-regulate SNAP-25 expression, thereby triggering synaptic vesicle exocytosis to regulate locomotion ability in C. elegans. Collectively, our findings may promote a comprehensive interpretation of the pharmacological mechanism of mercury sulfide on neuroprotective function.

Neuronal-specific TNFAIP1 ablation attenuates postoperative cognitive dysfunction via targeting SNAP25 for K48-linked ubiquitination

BackgroundSynaptosomal-associated protein 25 (SNAP25) exerts protective effects against postoperative cognitive dysfunction (POCD) by promoting PTEN-induced kinase 1 (PINK1)/Parkin-mediated mitophagy and repressing caspase-3/gasdermin E (GSDME)-mediated pyroptosis. However, the regulatory mechanisms of SNAP25 protein remain unclear.MethodsWe employed recombinant adeno-associated virus 9 (AAV9)-hSyn to knockdown tumor necrosis factor α-induced protein 1 (TNFAIP1) or SNAP25 and investigate the role of TNFAIP1 in POCD. Cognitive performance, hippocampal injury, mitophagy, and pyroptosis were assessed. Co-immunoprecipitation (co-IP) and ubiquitination assays were conducted to elucidate the mechanisms by which TNFAIP1 stabilizes SNAP25.ResultsOur results demonstrated that the ubiquitin ligase TNFAIP1 was upregulated in the hippocampus of mice following isoflurane (Iso) anesthesia and laparotomy. The N-terminal region (residues 1–96) of TNFAIP1 formed a conjugate with SNAP25, leading to lysine (K) 48-linked polyubiquitination of SNAP25 at K69. Silencing TNFAIP1 enhanced SH-SY5Y cell viability and conferred antioxidant, pro-mitophagy, and anti-pyroptosis properties in response to Iso and lipopolysaccharide (LPS) challenges. Conversely, TNFAIP1 overexpression reduced HT22 cell viability, increased reactive oxygen species (ROS) accumulation, impaired PINK1/Parkin-dependent mitophagy, and induced caspase-3/GSDME-dependent pyroptosis by suppressing SNAP25 expression. Neuron-specific knockdown of TNFAIP1 ameliorated POCD, restored mitophagy, and reduced pyroptosis, which was reversed by SNAP25 depletion.ConclusionsIn summary, our findings demonstrated that inhibiting TNFAIP1-mediated degradation of SNAP25 might be a promising therapeutic approach for mitigating postoperative cognitive decline.2d6TEYTUuUajqwtaSq8xEZVideo

A “toxic window” study on the hippocampal development of mice offspring exposed to azithromycin at different doses, courses, and time during pregnancy

BackgroundAzithromycin, one of the new-generation macrolides, is an effective medicine for the treatment of mycoplasma infection during pregnancy. Epidemiological studies have reported adverse pregnancy outcomes with prenatal azithromycin exposure (PAzE). However, the effect of PAzE on fetal hippocampal development is unclear. This study aimed to explore the effects and potential mechanism of PAzE-induced fetal hippocampal development at different doses, courses, and time. MethodPregnant mice were administered azithromycin by gavage at different doses (50, 100 or 200 mg/kg.d), different courses (gestational day (GD)15–17 for three consecutive days, or GD17 once a day) and different time (GD10-12, GD15-17). ResultsCompared with the control group, morphological development damage of the fetal hippocampus was observed in the PAzE group, with a dysbalance in neuronal proliferation and apoptosis, decreased expression of the neuronal-specific marker Snap25, NeuN, PSD95 and Map2, increased expression of the glial-specific marker Iba1, GFAP, and S-100β, and decreased expression of P2ry12. The PAzE-induced hippocampal developmental deficiency varied based on different doses, courses, and time, and the developmental toxicity was most significant in the late pregnancy, high dose, multi-course group (AZHT). The significant reduction of SOX2 and Wnt, which were related to regulation of neural progenitor cells (NPCs) proliferation in PAzE fetus compared with the control group indicated that the SOX2/Wnt signaling may be involved in PAzE-induced hippocampal developmental toxicity. ConclusionIn this study, PAzE was associated with hippocampal developmental toxicity in a variety of nerve cells. Hippocampal developmental toxicity due to azithromycin was most significant in the late pregnancy, high-dose (equivalent to maximum clinical dose) and multi-course group (AZHT). The findings provide an experimental and theoretical foundation for guiding the sensible use of medications during pregnancy and effectively assessing the risk of fetal hippocampal developmental toxicity.

Effect of caffeine on hippocampal memory and levels of gene expression in social isolation stress.

Caffeine (Cf) antagonizes the adenosine receptors and has neuroprotective properties. The effect of Cf has been seen on stress-induced deficits of cognitive. In this study, we have investigated the effect of Cf on learning and memory functions induced by social isolation (SI) stress. In the present study, 21-day-old Wistar albino male rats (n = 28) were divided into four groups: the control (C), the SI, the Cf, and the social isolation + caffeine (SICf). Cf (0.3 g/L) was added to the drinking water of the experimental animals for 4weeks. The learning and memory functions were assessed using the Morris Water Maze Test (MWMT). Following, was performed histopathological evaluation and determined hippocampal gene expression levels by RT-qPCR. According to MWMT findings, the time spent in the quadrant where the platform removed was decreased in the SI group compared with the C (p < 0.05). Histological evaluation showed morphological changes in SI by irregular appearance, cellular edema, and dark pycnotic appearance of nuclei in some neurons. However, it was observed that the histological structure of most of the neurons in the SICf group was similar to the C and Cf groups. Hippocampal SNAP25 expression was decreased in the Cf and SICf groups than in the C group (p < 0.05). The GFAP expression was increased in the SICf group than in the C group (p < 0.05). NR2A increased in the SI and SICf groups compared with C and Cf groups (p < 0.05). NR2B expression decreased in the Cf group compared with C and SI groups (p < 0.05). SI impaired spatial memory and causes morphological changes in adolescent rats, but this effect of isolation was not seen in Cf-treated animals. The effects of SI on NR2A, Cf on NR2B, and SNAP25 are remarkable. Here, we propose that the impaired effect of SI on spatial memory may be mediated by NR2A, but further studies are needed to explain this effect.

Ultrasound-assisted intravesical botulinum toxin A delivery attenuates acetic acid-induced bladder hyperactivity in rats.

Background: Intradetrusor injection of botulinum toxin A (BTX-A) is an effective treatment for overactive bladder (OAB). However, the occurrence of adverse events associated with BTX-A injection therapy hinders its acceptance among patients and its clinical promotion. Intravesical instillation of BTX-A offers a promising alternative to injection therapy for treating OAB. Nevertheless, due to the presence of the bladder permeability barrier (BPB) and the high molecular weight of BTX-A, direct instillation is unable to penetrate the bladder urothelium. Purpose: This study aims to investigate the safety and feasibility of ultrasound-assisted intravesical delivery of BTX-A and its potential benefits in a rat model of bladder hyperactivity induced by acetic acid instillation. Methods: Hengli BTX-A and microbubbles (MB) were mixed and prepared as a novel complex. The size distribution and zeta potentials of the complex were measured. On day 1, rats' bladders were instilled with 1mL of saline, BTX-A (20U in 1mL), MB, or MB-BTX-A (20U in 1mL) complex with or without ultrasound (US) exposure (1MHz, 1.5W/cm2, 50% duty cycle, sonication for 10s with a 10-s pause for a total of 10min). The instillations were maintained for 30min. After 7days, cystometry was performed by filling the bladder with saline and 0.3% acetic acid (AA). Bladders were collected, weighed, and processed for immunoblotting, enzyme-linked immunosorbent assay (ELISA), histologic, and immunofluorescence analyses. Expression and distribution of SNAP-25 and SNAP-23 were assessed using Western blot and immunofluorescence. Calcitonin gene-related peptide (CGRP) in the bladder was detected using ELISA. Results: Intercontraction intervals (ICI) decreased by 72.99%, 76.16%, and 73.96% in rats pretreated with saline, BTX-A, and US + MB, respectively. However, rats treated with US + MB + BTX-A showed a significantly reduced response to AA instillation (57.31% decrease in ICI) without affecting amplitude, baseline pressure, or threshold pressure. Rats treated with US + MB + BTX-A exhibited increased cleavage of SNAP-25 and CGRP expression compared to the control group. Conclusion: Ultrasound-assisted intravesical delivery of BTX-A, with the assistance of MB cavitation, led to cleavage of SNAP-25, inhibition of calcitonin gene-related peptide release from afferent nerve terminals, and amelioration of acetic acid-induced bladder hyperactivity. These results support ultrasound-assisted intravesical delivery as an efficient non-injection method for administering BTX-A.

Boldine modulates glial transcription and functional recovery in a murine model of contusion spinal cord injury.

Membrane channels such as those formed by connexins (Cx) and P2X7 receptors (P2X7R) are permeable to calcium ions and other small molecules such as adenosine triphosphate (ATP) and glutamate. Release of ATP and glutamate through these channels is a key mechanism driving tissue response to traumas such as spinal cord injury (SCI). Boldine, an alkaloid isolated from the Chilean boldo tree, blocks both Cx and Panx1 hemichannels (HCs). To test if boldine could improve function after SCI, boldine or vehicle was administered to treat mice with a moderate severity contusion-induced SCI. Boldine led to greater spared white matter and increased locomotor function as determined by the Basso Mouse Scale and horizontal ladder rung walk tests. Boldine treatment reduced immunostaining for markers of activated microglia (Iba1) and astrocytic (GFAP) markers while increasing that for axon growth and neuroplasticity (GAP-43). Cell culture studies demonstrated that boldine blocked glial HC, specifically Cx26 and Cx30, in cultured astrocytes and blocked calcium entry through activated P2X7R. RT-qPCR studies showed that boldine treatment reduced expression of the chemokine Ccl2, cytokine IL-6 and microglial gene CD68, while increasing expression of the neurotransmission genes Snap25 and Grin2b, and Gap-43. Bulk RNA sequencing revealed that boldine modulated a large number of genes involved in neurotransmission in spinal cord tissue just caudal from the lesion epicenter at 14 days after SCI. Numbers of genes regulated by boldine was much lower at 28 days after injury. These results indicate that boldine treatment ameliorates injury and spares tissue to increase locomotor function.

PIMT Controls Insulin Synthesis and Secretion through PDX1.

Pancreatic beta cell function is an important component of glucose homeostasis. Here, we investigated the function of PIMT (PRIP-interacting protein with methyl transferase domain), a transcriptional co-activator binding protein, in the pancreatic beta cells. We observed that the protein levels of PIMT, along with key beta cell markers such as PDX1 (pancreatic and duodenal homeobox 1) and MafA (MAF bZIP transcription factor A), were reduced in the beta cells exposed to hyperglycemic and hyperlipidemic conditions. Consistently, PIMT levels were reduced in the pancreatic islets isolated from high fat diet (HFD)-fed mice. The RNA sequencing analysis of PIMT knockdown beta cells identified that the expression of key genes involved in insulin secretory pathway, Ins1 (insulin 1), Ins2 (insulin 2), Kcnj11 (potassium inwardly-rectifying channel, subfamily J, member 11), Kcnn1 (potassium calcium-activated channel subfamily N member 1), Rab3a (member RAS oncogene family), Gnas (GNAS complex locus), Syt13 (synaptotagmin 13), Pax6 (paired box 6), Klf11 (Kruppel-Like Factor 11), and Nr4a1 (nuclear receptor subfamily 4, group A, member 1) was attenuated due to PIMT depletion. PIMT ablation in the pancreatic beta cells and in the rat pancreatic islets led to decreased protein levels of PDX1 and MafA, resulting in the reduction in glucose-stimulated insulin secretion (GSIS). The results from the immunoprecipitation and ChIP experiments revealed the interaction of PIMT with PDX1 and MafA, and its recruitment to the insulin promoter, respectively. Importantly, PIMT ablation in beta cells resulted in the nuclear translocation of insulin. Surprisingly, forced expression of PIMT in beta cells abrogated GSIS, while Ins1 and Ins2 transcript levels were subtly enhanced. On the other hand, the expression of genes, PRIP/Asc2/Ncoa6 (nuclear receptor coactivator 6), Pax6, Kcnj11, Syt13, Stxbp1 (syntaxin binding protein 1), and Snap25 (synaptosome associated protein 25) associated with insulin secretion, was significantly reduced, providing an explanation for the decreased GSIS upon PIMT overexpression. Our findings highlight the importance of PIMT in the regulation of insulin synthesis and secretion in beta cells.

Analgesic Effect of Perineural Injection of BoNT/A on Neuropathic Pain Induced by Chronic Constriction Injury of Sciatic Nerve in Rats.

This study was designed to investigate the analgesic effect of perineural injection of BoNT/A on neuropathic pain induced by sciatic nerve chronic constriction injury (CCI) and possible mechanisms. SD rats were randomly divided into Sham group, CCI group and BoNT/A group. Paw mechanical withdrawal threshold (pMWT) and paw thermal withdrawal latency (pTWL) of each group were detected at different time points after surgery. The expression of myelin markers, autophagy markers and NLRP3 inflammasome-related molecules in injured sciatic nerves were examined at 12days after surgery. Moreover, C-fiber evoked potential in spinal dorsal horn was recorded. The expression of SNAP-25, neuroinflammation and synaptic plasticity in spinal dorsal horn of each group were examined. Then rats treated with BoNT/A were randomly divided into DMSO group and Wnt agonist group to further explore the regulatory effect of BoNT/A on Wnt pathway. We found that pMWT and pTWL of ipsilateral paw were significantly decreased in CCI group compared with Sham group, which could be improved by perineural injection of BoNT/A at days 7, 9 and 12 after surgery. The peripheral analgesic mechanisms of perineural injection of BoNT/A might be related to the protective effect on myelin sheath by inhibiting NLRP3 inflammasome and promoting autophagy flow, while the central analgesic mechanisms might be associated with inhibition of neuroinflammation and synaptic plasticity in spinal dorsal horn due to inhibiting SNAP-25 and Wnt pathway. As a new route of administration, perineural injection of BoNT/A can relieve CCI induced neuropathic pain probablyvia both peripheral andcentral analgesic mechanisms.

Molecular changes underlying decay of sensory responses and enhanced seizure propensity in peritumoral neurons.

Glioblastoma growth impacts on the structure and physiology of peritumoral neuronal networks, altering the activity of pyramidal neurons which drives further tumor progression. It is therefore of paramount importance to identify glioma-induced changes in pyramidal neurons, since they represent a key therapeutic target. We longitudinal monitored visual evoked potentials after the orthotopic implant of murine glioma cells into the mouse occipital cortex. With laser microdissection, we analyzed layer II-III pyramidal neurons molecular profile and with local field potentials recordings we evaluated the propensity to seizures in glioma-bearing animals with respect to control mice. We determine the time course of neuronal dysfunction of glioma-bearing mice and we identify a symptomatic stage, based on the decay of visual response. At that time point, we microdissect layer II-III pyramidal neurons and evaluate the expression of a panel of genes involved in synaptic transmission and neuronal excitability. Compared to the control group, peritumoral neurons show a decrease in the expression of the SNARE complex gene SNAP25 and the alpha1 subunit of the GABA-A receptor. No significant changes are detected in glutamatergic (ie, AMPA or NMDA receptor subunit) markers. Further reduction of GABA-A signaling by delivery of a benzodiazepine inverse agonist, DMCM (methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) precipitates seizures in 2 mouse models of tumor-bearing mice. These studies reveal novel molecular changes that occur in the principal cells of the tumor-adjacent zone. These modifications may be therapeutically targeted to ameliorate patients' quality of life.

Sex-specific effects of SNAP-25 genotype on verbal memory and Alzheimer's disease biomarkers in clinically normal older adults.

We tested sex-dependent associations of variation in the SNAP-25 gene, which encodes a presynaptic protein involved in hippocampal plasticity and memory, on cognitive and Alzheimer's disease (AD) neuroimaging outcomes in clinically normal adults. Participants were genotyped for SNAP-25 rs1051312 (T>C; SNAP-25 expression: C-allele>T/T). In a discovery cohort (N=311), we tested the sex by SNAP-25 variant interaction on cognition, Aβ-PET positivity, and temporal lobe volumes. Cognitive models were replicated in an independent cohort (N=82). In the discovery cohort, C-allele carriers exhibited better verbal memory and language, lower Aβ-PET positivity rates, and larger temporal volumes than T/T homozygotes among females, but not males. Larger temporal volumes related to better verbal memory only in C-carrier females. The female-specific C-allele verbal memory advantage was evidenced in the replication cohort. In females, genetic variation in SNAP-25 is associated with resistance to amyloid plaque formation and may support verbal memory through fortification of temporal lobe architecture. The SNAP-25 rs1051312 (T>C) C-allele results in higher basal SNAP-25 expression. C-allele carriers had better verbal memory in clinically normal women, but not men. Female C-carriers had higher temporal lobe volumes, which predicted verbal memory. Female C-carriers also exhibited the lowest rates of amyloid-beta PET positivity. The SNAP-25 gene may influence female-specific resistance to Alzheimer's disease (AD).

MiR-23a-3p and miR-181a-5p modulate SNAP-25 expression.

SNAP-25 protein is a key protein of the SNARE complex that is involved in synaptic vesicles fusion with plasma membranes and neurotransmitter release, playing a fundamental role in neural plasticity. Recently the concentration of three specific miRNAs-miR-27b-3p, miR-181a-5p and miR-23a-3p -was found to be associated with a specific SNAP-25 polymorphism (rs363050). in silico analysis showed that all the three miRNAs target SNAP-25, but the effect of the interaction between these miRNAs and the 3'UTR of SNAP-25 mRNA is currently unknown. For this reason, we verified in vitro whether miR-27b-3p, miR-181a-5p and miR-23a-3p modulate SNAP-25 gene and protein expression. Initial experiments using miRNAs-co-transfected Vero cells and SNAP-25 3'UTR luciferase reporter plasmids showed that miR-181a-5p (p≤0.01) and miR-23a-3p (p<0.05), but not miR-27b-3p, modulate the luciferase signal, indicating that these two miRNAs bind the SNAP-25 3'UTR. Results obtained using human oligodendroglial cell line (MO3.13) transfected with miR-181a-5p or miR-27b-3p confirmed that miR-181a-5p and miR-23a-3p regulate SNAP-25 gene and protein expression. Interestingly, the two miRNAs modulate in an opposite way SNAP-25, as miR-181a-5p significantly increases (p<0.0005), whereas miR-23a-3p decreases (p<0.0005) its expression. These results for the first time describe the ability of miR-181a-5p and miR-23a-3p to modulate SNAP-25 expression, suggesting their possible use as biomarkers or as therapeutical targets for diseases in which SNAP-25 expression is altered.